The present invention relates to proteins having a hemolytic activity and genes encoding thereof. More specifically, the present invention relates to novel proteins having the hemolytic activity, a process for producing and the use of the same.
The sting injury by the jellyfish in sea bathing has occurred in various parts of the world. The sting injury by Carybdea rastonii or Physalia physalis has also occurred frequently in Japan every year in the season of sea bathing of the summertime. The degree of the symptom by sting differs by species of a jellyfish and the individual differences of patients. The first symptom is dermotoses, such as pain, flare, papule, vesicle and so on in the sting site. In a serious illness, patients may die with generating of hemorrhagic maculae and the necrosis, and also constitutional symptom, such as headache, high fever, nausea, dyspnea, and the fluctuation of a pulse.
Although such sting injury is occurring frequently, the determination and pharmacological properties of the toxic components of jellyfish have not been studied intensively.
Therefore, the development of medicines for treatment of the sting by the jellyfish is hardly performed before the present invention.
The studies on the toxic components of Carybdea rastonii have reported by Sato et al., and they found that there are some active substances having physiological activities, such as hemolysis, platelet agglutination, mast cell degranulation, the vessel smoothness muscle contraction, the dermal necrosis, the heart poison and the fatality in the crude extract fractions from the freeze-dried tentacle of Carybdea rastonii. They also examined on the platelet agglutination effect and vessel smoothness muscle contraction effect of the toxic component (Akihiko Sato, xe2x80x9cResearch on the toxic component of Carybdea rastoniixe2x80x9d, The Journal of the Ochanomizu Medico-dental Society, vol. 33, No. 2, 131-151, June, 1985).
On the one hand, since the poison from the nematocyst of a jellyfish was a non-dialyzable high polymer and deactivated by treatment with acid or alkali, or by heating processing, organic solvent processing, protease processing, etc., it was thought that the main components of this poison were proteins.
Moreover, the purification of the protein toxin derived from a jellyfish has also been tried; however, the isolation and the purification of the active components maintaining the hemolytic activity were not performed since the toxin of a jellyfish itself was very easy to be deactivated. Therefore, the physical and chemical properties of the toxin from jellyfish were not known up to now.
The detailed studies on the toxic component of a jellyfish is very important for the development of drugs applying their various physiological activities, in particular, specific hemolytic activity and the platelet agglutination effect.
Therefore, the problems to be solved by the present invention is providing an approach to development of the drugs for treatment of the sting injury by the jellyfish by means of isolating the proteins or peptides having as potent hemolytic activity as possible, in the state where the physiologic activity is retained. The present invention further provides the approach to study similarities on embryology or structure, and the species specificity of the protein having hemolytic activity to evaluate the structure-activity relationship thereof.
The inventors extensively performed the research for isolating the proteins having the hemolytic activity from the nematocyst of Carybdea rastonii using the hemolytic activity as the parameter, while retaining these hemolytic activities. As the result, they found out the process for isolating and purifying the proteins retaining hemolytic activities, and found that the protein from Carybdea rastonii had the partial chemical structure consisting the following amino acid sequences (1)-(3), and the molecular weight of about 50,000 Da (determined by SDS gel electrophoresis).
Amino acid sequence (1):
Gly-Glu-Ile-Gln-Thr-Lys-Pro-Asp-Arg-Val-Gly-Gln-Ala-Thr (SEQ ID NO:1)
Amino acid sequence (2):
Gly-Asn-Ala-Glu-His-Val-Ala-Ser-Ala-Val-Glu-Asn-Ala-Asn-Arg-Val-Asn-Lys (SEQ ID NO:2)
Amino acid sequence (3):
Met-Ser-Asp-Gly-Phe-Tyr-Thr-Met-Glu-Asn-Ser-Asp-Arg-Arg-Lys (SEQ ID NO:3)
(wherein, an amino acid residue is written by the 3 letters notation defined by IUPAC and IUB)
Furthermore, they prepared the primers based on their partial chemical structures of the protein, and analyzed the gene sequence of about 1,000 base pair of said protein by conducting the RT-PCR on the total RNA prepared from the tentacle of Carybdea rastonii by using these primers. Consequently, they further determined the full primary amino acid sequence of the hemolytic active protein of Carybdea rastonii by means of analyzing the gene sequence at the 5xe2x80x2-end and 3xe2x80x2-end using the 5xe2x80x2 RACE method and 3xe2x80x2 RACE method.
Therefore, one embodiment of the present invention provides the specific protein having above-mentioned physiological, physical and chemical properties and represented by the amino acid (SEQ ID NO:5) or the amino acid sequence thereof partially modified by the deletion or substitution of amino acid, and/or the amino acid sequence thereof partially modified by the deletion or substitution of amino acid further one or more amino acids are added.
Another embodiment of the present invention also provides the process for preparing such proteins.
Furthermore, another embodiment provides the gene encoding such proteins, the process for preparing the specific proteins using the gene, and the drugs or the pesticides using the same.
The present invention further provides the pharmaceutical compositions or the pesticides containing the proteins using these properties, particularly, the pharmaceutical compositions having the platelet agglutination effect etc.
Moreover, since a specific antibody can also be obtained from this hemolytic active protein according to a conventional method (Cell Technology, separate volume, xe2x80x9cExperimental protocol of antipeptide antibodyxe2x80x9d, Shujunsha Co.), the present invention also provides the pharmaceutical compositions containing said antibody.
The isolation and purification of the proteins having the specific physiological activity provided by the present invention can specifically be performed as follows. For example, the ultrasonication of the nematocyst of Carybdea rastonii is carried out in phosphoric acid buffer solution, and then supernatants are collected by the centrifugal separation to obtain a crude extract. The object proteins can be separated and purified by subjecting this crude extract to ion exchange high performance liquid chromatography using TSK-GEL (Toso Co.), and the gel filtration high performance liquid chromatography with Superdex-75 (Pharmacia Co.).
The structure of the protein provided according to the present invention obtained in this way can be determined by combining the analysis procedure of the amino acid sequence by the selective degradation using the enzyme, and the analysis procedure of a gene sequence using the PCR method etc. For example, the amino acid sequence can be determined by processing the protein separated and purified as mentioned above with a lysylendopeptidase, fractionating the fragment using a high performance liquid chromatography, and analyzing it using an amino acid sequencer etc. Next, the gene sequence of the proteins can be determined by RT-PCR method etc. using the primers prepared on the basis of the amino acid sequence. Finally, the full primary amino acid sequence of the proteins can be clarified by determining the amino acid sequence on the basis of the gene sequence.
It was confirmed by such analysis that the protein provided according to the present invention has the molecular weight of about 50,000 Da (measured by SDS gel electrophoresis), and the partial amino acid sequences have the above-mentioned amino acid sequences (1) to (3).
As a result of homology search on the partial amino acid sequences, the homology between the protein of the present invention and the known proteins was very low. Therefore, it was suggested that the protein of the present invention having the hemolytic activity is completely novel protein, which is not similar to the known proteins.
Next, the determination of the gene sequence of about 1,000 base pairs by performing RT-PCR to total RNA prepared from the tentacle of Carybdea rastonii using the primers prepared on the basis of the partial amino acid sequence, and the determination of the gene sequences of the 5xe2x80x2-end and the 3xe2x80x2-end using the 5xe2x80x2 RACE method and 3xe2x80x2 RACE method were performed. Consequently, it is concluded that the hemolytic active protein of Carybdea rastonii has the full primary amino acid sequence represented by (SEQ ID NO:5) and the gene encoding thereof has the base sequence represented by (SEQ ID NO:4).
The result of the homology search on these full primary amino acid sequences exhibited that the homology between the protein and the known proteins was low.
The method for preparing the specific protein of the present invention by separation and purification is characterized in retaining the hemolytic activity. For example, the separation and the purification in the state of retaining such hemolytic activity are attained by performing the processing such as ultrasonication using the above-mentioned phosphoric acid buffer solution or various high performance liquid chromatography in 10 mM phosphoric acid buffer solution (pH 6.0) containing above 0.1 M NaCl, preferably above 0.3 M, and more preferably above 0.5 M, at below 10xc2x0 C., preferably below 5xc2x0 C.
Therefore, the present invention also provides the method for preparing the protein by extracting and purifying them from the nematocyst of the Carybdea rastonii in the state of retaining the physiological activity.
The specific protein of the present invention also can be prepared by the gene recombination method. Preparation by the gene recombination method can be performed according to a conventional method. For example, it can be obtained by preparing the vector integrated with the gene represented by (SEQ ID NO:4), transforming a host cell by the vector, incubating or growing the host cell, and isolating and purifying the proteins having hemolytic activity of interest from the host cell or culture solution.
Since the protein provided according to the present invention has a hemolytic activity, for example, it may be used for the medicaments having the platelet agglutination effect and for the reagents for research on a hemolysis. Furthermore, it provides the new approach for the development of drugs, such as a drug for treating the sting by the jellyfish, and development of pesticides, such as an insecticide, using the hemolytic activity.